Protein S-palmitoylation is a post-translational modification (PTM) where a fatty acyl moiety (saturated 16C palmitate) is linked via a thioester bond to a cysteine residue on target proteins. Unlike myristoylation and prenylation, which are irreversible processes, palmitoylation is dynamic and reversible. Enzymes termed protein acyltransferases (PATs) attach the palmitate group to proteins, while acyl-protein thioesterases (APTs) remove the modification by hydrolysis of the thioester bond. Despite being a PTM that regulates a range of dynamic process including cell signaling, cell division and synapse formation, very few examples of reversibly palmitoylated proteins have been documented, reflecting the general lack of tools available for studying this dynamic process. We recently identified a class of compounds that enhance the process of host cell invasion by the parasite pathogen Toxoplasma gondii. We have determined that these compounds function by binding and inhibiting the parasite homolog of human acyl-protein thioesterase 1 (APT1), a hydrolase involved in depalmitoylation of a range of signaling proteins including Ras, eNOS and G proteins. Our compounds directly block the function of this enzyme, resulting in accumulation of palmitoylated substrates and alteration of parasite motility and organelle secretion. In addition, homology search and recent reports indicate that parasites may express three additional acyl-protein thioesterases. We hypothesize that reversible palmitoylation is a key regulatory process used by T. gondii and likely other human pathogens to regulate important processes and that understanding how regulated removal of palmitate groups on specific substrates will shed light on pathways that can be disrupted for therapeutic gain. Therefore, we propose to 1) determine the repertoire of depalmitoylating enzymes in T. gondii and develop small molecule inhibitors to study their function 2) Use a chemical proteomics strategy to identify candidate protein substrates regulated by dynamic palmitoylation and 3) develop chemical tools to validate the importance of specific depalmitoylation events. This proposal makes use of diverse chemical, biochemical, proteomic and cell biological methods to accomplish these aims.